1. Field of the Invention
This invention relates to gene expression and more particularly to a chromosomal expression vector based on the bacteriophage Mu.
2. Related Art
Over-expression of heterologous genes in E. coli has become a routine method as a first step for obtaining large amounts of pure protein. The heterologous gene is usually cloned downstream of a strong E. coli promoter on a multicopy plasmid. The amount of heterologous protein produced from such a system can account for as much as 40% of total cell protein. Although this system has been extremely useful, it does have some drawbacks. The major one is the instability of the multicopy plasmid. There is no system in place to ensure inheritance of most high copy number plasmids used in recombinant expression systems. If the copy number is high enough, mathematical models suggest that plasmid loss should not be a problem (Summers, 1991). However, high copy number recombinant plasmids are often not as stable as these models would predict (Summers, 1991). Any plasmidless cells that do appear often out grow those containing plasmids eventually leading to the loss of plasmid from nearly the entire population (Zund and Lebek, 1980).
The problem of plasmid instability is compounded when large volumes of cells are to be grown for production. This problem occurs during the large number of doublings in a chemostat for continuous culture or during subculturings to obtain an inoculum of appropriate size for a batch process. One common way to maintain the plasmid is to exert selective pressure, usually by the presence of an antibiotic. The inclusion of an antibiotic in the culture medium for large scale protein production can be a problem in terms of high cost and disposal. Furthermore, antibiotics such as .beta.-lactams that are broken down can not always be used successfully to maintain the plasmid.
Several genetic methods have been described to overcome the problem of plasmid instability. Partition sequences from lower copy number plasmids have been included such as par (Skogman, et al. 1983), cer (Summers and Sherrat, 1984), and hok/sok (Gerdes, 1988). Although these systems do help stabilize the plasmid there is still plasmid loss when cells are grown in continuous culture. Another option is to include an essential gene on the plasmid such as ssb (Porter, et al., 1990). Any cells that lose the plasmid will die and not be able to overtake the population. This system has the disadvantage that the expression vector must replace a resident plasmid containing the essential gene.
A second factor leading to plasmid instability can be synthesis of a heterologous protein that is deleterious to the cell. In this case, any cells not making the protein will out compete those making the protein resulting in a plasmidless population. One way to circumvent this problem is to have expression of the gene very tightly regulated using promoters such as P.sub.lac. However, it should be noted that it is unlikely that any promoter is ever entirely shut off and whatever low level transcription occurs will be multiplied by the high copy number.
The problems of instability and product lethality may be circumvented by having the heterologous gene present as a single copy on the chromosome prior to induction and present at many copies post-induction. Systems that use this switch in nature are lysogenic bacteriophage such as lambda. During the lysogenic state the phage is integrated as a single copy and is segregated with the chromosome at cell division ensuring inheritance. Upon induction of the lytic cycle the lambda genome is excised from the chromosome and replicates extrachromosomally resulting in as many as 100 copies. Lambda variants have been used to overexpress a limited number of genes (Padukone, et al., 1990, Nakano and Masuda, 1982 and Panasenko, et al., 1977). This system is not more widely used because the large genome size of lambda makes it cumbersome to work with and in some cases the cells lyse following induction.
The present invention is based on a different lysogenic bacteriophage, Mu. Mu differs from lambda in that it never replicates extrachromosomally. Upon induction of the lytic cycle Mu undergoes multiple rounds of replicative transposition resulting in as many as 100 copies of Mu DNA integrated into the host chromosome. All the replicative transposition functions including regulation of the switch from lysogenic to lytic phase are contained within a 4.5 kbp fragment on the left end of Mu thus eliminating the size problem encountered with lambda. Variants of Mu have been constructed that are capable of replicative transposition but are deleted for middle and late functions and therefore can not make phage particles. One such derivative, Mud4041, includes the Km-resistance antibiotic marker, allowing for easy selection of lysogens (Castilho, et al., 1984). To create a chromosomal expression vector, the Mu middle promoter, Pm, was introduced to regulate expression of heterologous genes. This promoter was chosen since it is induced following the start of Mu replication (Marrs and Howe, 1990, Stoddard and Howe, 1989). Induction of the promoter also requires the transactivator mor (Mathee and Howe, 1990). The Mu repressor of the lytic cycle, cts62, present on Mud4041 is temperature sensitive, therefore, by shifting the culture from 30.degree. to 42.degree. C., both replication of Mu and transcription from Pm will be induced. Using this system a number of heterologous proteins were made at 5 to 20% of total cell protein.